Lecture 8 - Protein function pt. 2

what does n in the Hill equation mean?

n is the Hill Coefficient - the degree of cooperativity or interaction between ligand-binding sites in the protein

• n < 1 (negative cooperativity) - binding of ligand at one site decreases affinity of other binding sites in the protein for that ligand

• n = 1 (no cooperativity) - binding sites act independently of each other

• n > 1 (positive cooperativity) - binding of ligand at one site increases affinity of other binding sites in the protein for that ligand

  • value represents number of binding sites that are fully cooperating

• n is calculated from the Hill plots

what is the Hill coefficient

slope of the line on the Hill plot

what are the slopes of Mb and Hb on the hill plot

• Mb = 1

  • therefore no cooperativity

• Hb = 3

  • therefore high cooperativity

asymptotes on Hill plot

• lower asymptote gives p50 of 1st O2 bound

  • pretty high since Hb is in T state when no o2 is bound

    • Hb has poor binding affinity with no O2 bound

• last O2 bound has p50 = 0.3 torr (which is lower)

  • meaning that due to cooperativity, Hb has very high affinity for O2 for the last O2 binding site

myoglobin vs. hemoglobin

• myoglobin

  • monomeric, one heme

  • hyperbolic O2 binding curve

  • p50 = 2.8 torr

• hemoglobin

  • tetrameric, four hemes (exhibit cooperative binding)

  • sigmoidal O2 binding curve

  • p50 = 26 torr

physiological roles of Mb vs. Hb

• Hemoglobin - transportation of O2

  • pO2 in lungs is ~100 torr (Y02 = 0.9), in tissues pO2 ~ 30 torr (YO2 = 0.5)

    • Hb binds O2 in the lungs and releases it in tissue

• Myoglobin - storage of O2 (delivered by Hb) in muscle tissues

  • Po2 in tissue is ~ 30 torr (YO2 = 0.9)

    • can be much lower in metabolically active tissue

    • Mb binds O2 delivered by Hb and releases it when tissue O2 lvls decrease

why cant Mb perform transportation like Hb

because even though it can bind O2 in the lungs, it cannot release it in the tissue

• no cooperative O2 binding in Mb

describe series of events that happen after O2 binding on one subunit of Hb causes a shift from the T to the R state

• central cavity of Hb changes size

  • gets narrower as it goes from deoxyhemoglobin (T state) to oxyhemoglobin (R state)

• BPG is releases out of the molecule

• release of H+ and Cl-

allosteric protein

protein in which binding of a ligand to one site affects binding of a ligand to a different site

• ex. hemoglobin

  • cooperative O2 binding

homotropic vs. heterotropic allosterism

• homotropic allosterism - allosteric interaction using 2 of the same ligands

• heterotropic allosterism - allosteric interaction using 2 different types of ligands

homotropic vs. heterotropic allosteric effectors of Hb

• homotropic allosteric effector of Hb - O2

• heterotropic allosteric effectors of Hb - H+, Cl-, CO2 (HCO3-), D-2,3-biphosphoglycerate (BPG)

allosteric effects in hemoglobin

stabilize the T state making Hb (O2) unload the oxygen

allosteric protein structure

• often have regions in protein structure with lower stability

  • when ligand binds, it may introduce interactions that stabilize this region

    • affects structure of adjacent regions and changes their affinity for binding other ligands

• flexibility in peptide structure allows for conformational changes

  • allows for cooperative binding effects

Bohr effect (and mechanism)

allosteric regulation of Hb binding O2

• increase in H+ (which decreases pH) OR increase in CO2 causes increase in O2 binding affinity of Hb

• conformational change from T state to R state during O2 binding causes release of ~0.6 H+ for every O2 molecule bounds

  • caused by shifts in pKa values of aa residues

effect of increasing vs. decreasing pH

• decreasing pH

  • increase in H+ shifts deoxyhemoglobin / oxyhemoglobin (Hb / HbO2) equilibrium towards releasing O2 (T state)

• increasing pH

  • decrease in H+ shifts equilibrium towards binding O2

actively working muscle and O2

• actively working muscle produces H+ and lowers pH

• higher [H+] leads to Hb(O2) releasing O2 that the muscle needs

How does CO2 affect O2 binding in hemoglobin

through formation of carbamates with the amino groups at the N-termini of the peptide subunits

• T state of Hb binds more CO2 as carbamates than the R state does

• increasing pCO2 shifts Hb / HbO2 equilibrium towards releasing O2

• decreasing pCO2 shifts equilibrium towards binding O2

actively working muscle and CO2

actively working muscles makes CO2 through respiration

• increase in [CO2] also helps Hb unload needed O2

carbonic anhydrase

enzyme in RBCs that catalyzes conversion of CO2 and H2O into bicarbonate and protons

• catalyzes reaction in Muscle Tissue: CO2 + H2O → HCO3- + H+

• catalyzes the reverse reaction in lungs

  • allows CO2 gas to be regenerated and eliminated by exhalation

2 main ways CO2 is transported by Hb and red blood cells

• as carbamates

• as bicarbonate

  • since it cant cross RBC membrane, CO2 is transported in RBC in this form

    • RBC acts as transport vehicles because of this

how does Hb carry CO2 and H+ in the bloodstream (look at diagram for help)

• when you inhale (pO2 = 100 torr), Hb binds oxygen and carries it from bloodstream to muscle tissue (pO2 = 20 torr)

  • Mb binds to O2 since at this pO2, Hb affinity for O2 drops

• when muscles become intensely active, pO2 drops even lower (lower than p50 = 2.8 torr) and Mb is forced to release O2

  • O2 gets used where muscles need it

• Respiration produces CO2 and H2O

• carbonic anhydrase converts it into bicarbonate (HCO3-) and H+

  • so that CO2 can be transported by red blood cells back to the lungs

  • H+ is carried by hemoglobin

• when approaching the lungs, the reverse reaction occurs

  • regenerates CO2 to be exhaled out of the lungs

Chloride (Cl-) ions

Cl- are also released by conformational change of Hb from T to R state

• Cl- acts as allosteric modifier with similar effect as H+

BPG

D-2,3-biphosphoglycerate (BPG)

• allosteric effector of Hb in mammals

• concentrated in red blood cells

• shifts the Hb / HbO2 equilibrium towards releasing O2

How does BPG work as an allosteric effector ?

• forms attractive ionic interactions with 8 cationic groups in central cavity of deoxyhemoglobin

  • from each of the two B units

    • Lys82 (Lys EF6)

    • His143 (His H21)

    • His2 (His NA2)

    • N-terminal amino group of Val1 (Val NA1)

How does BPG change Hemoglobin O2 binding

• BPG binding shifts Hb / HbO2 equilibrium towards releasing O2

• in absence of BPG (“stripped Hb”), Hb has significantly higher affinity for O2

  • lower p50 value

• BPG decreases affinity of Hb for O2 so that O2 will be released in tissue (pO2 = ~30 torr)

fetal hemoglobin (a2Y2)

• instead of B subunits, they have Y subunits (variation of B subunit)

  • His143 (HisH21) is replaced with a Ser residue

• has higher O2 binding affinity than adult Hb

  • facilitate transfer of O2 between mother and fetus via the placenta

why does fetal Hemoglobin have higher O2 binding affinity than adult hemoglobin

the uncharged Ser R group cant interact with BPG

• detracts BPG’s ability to stabilize T state

  • therefore decreases effect of BPG on decreasing O2 affinity of Hb

How does BPG work in high-altitude adaptation?

• pO2 decreases with increasing altitude meaning insufficient O2 is reaching tissues (since not enough O2 is being released by Hb)

• RBCs rapidly change level of BPG production to adjust the o2 affinity of Hb to meet the changing conditions

  • goes from p50 = 26 torr (normal BPG concentration) to p50 = 31 torr (high BPG concentration)

• higher BPG concentration in RBCs (due to higher altitude) causes decrease in affinity of Hb for O2

  • thus increases amount of O2 released in tissues

what two models help explain cooperative binding in multisubunit proteins

• symmetry (MWC, concerted) model

• sequential (KNF) model

• both models assume that subunits are symmetrical, have only 2 conformations (R and T) which are in equilibrium

what do the symmetry and sequential models both assume ?

• both assume that subunits are symmetrical

• only have 2 conformations (R and T) which are in equilibrium

• have a ligand which can bind either R or T states

  • but affinities (of T and R state) for the ligand are different

Symmetry (MWC, concerted) model of allosterism

all subunits are postulated to be in same conformation at the same time (all R or all T)

• if ligand binds more tightly in R state than in T state, then binding of ligand will shift equilibrium towards R state

  • this increases affinity of unbound subunits for ligand

  • therefore cooperative binding

sequential (KNF) model of allosterism

any given subunit can be either R or T state

• binding of ligand to subunit induces change of that subunit from R to T

• cooperative interactions promote binding of ligands to neighbouring subunits so that they also transform

sickle cell anemia

inherited disease caused by DNA mutation which causes substitution of Glu6 (Glu A3) in the B subunits for a Val residue

• hemoglobin S has 2 fewer negative charges than wild type hemoglobin

  • is more hydrophobic

• causes hemolytic anemia

sickle cell anemia

inherited disease caused by DNA mutation which causes substitution of Glu6 (Glu A3) in the B subunits for a Val residue

• hemoglobin S has 2 fewer negative charges than wild type hemoglobin

  • is more hydrophobic

• causes hemolytic anemia

how does a single aa substitution in Hb cause sickle cell anemia?

• mutant Val6 residue on deoxyhemoglobin S forms hydrophobic interactions with a hydrophobic pocket on another deoxyhemoglobin S molecule

• these polymers precipitate

  • causes RBCs to sickle which impede their flow through capillaries

    • decreases O2 delivery to tissue and results in tissue death